Gene cloning, nucleotide sequencing, and purification and characterization of the low-specificity L-threonine aldolase from Pseudomonas sp. strain NCIMB 10558.
A low-specificity L-threonine aldolase (L-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia coli cells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5'-phosphate as a coenzyme, is strictly L specific at the alpha position, whereas it cannot distinguish between threo and erythro forms at the beta position. In addition to threonine, the enzyme also acts on various other L-beta-hydroxy-alpha-amino acids, including L-beta-3,4-dihydroxyphenylserine, L-beta-3,4-methylenedioxyphenylserine, and L-beta-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificity L-TA from Saccharomyces cerevisiae, L-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity L-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of the L-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5'-phosphate of the enzyme to catalyze the reversible aldol reaction.
Liu
J Q
JQ
Laboratory of Biocatalytic Chemistry, Toyama Prefectural University, Japan.
Ito
S
S
Dairi
T
T
Itoh
N
N
Kataoka
M
M
Shimizu
S
S
Yamada
H
H
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D012696:Glycine Hydroxymethyltransferase